(1) Field of the Invention
The present invention relates to triggerable stable 1,2-dioxetanes which have the ability to bind to organic and biological molecules for use in assays and the like. In particular, the present invention relates to 1,2-dioxetanes which have an ability to react with biological materials without materially altering their essential function.
(2) PRIOR ART
1. Chemical Triggering of Stabilized 1,2-Dioxetanes. Thermally stable dioxetanes which can be triggered by chemical and enzymatic processes to generate chemiluminescence on demand have recently been discovered (A. P. Schaap, patent application Ser. No. 887,139, filed Jul. 17, 1986; A. P. Schaap, R. S. Handley, and B. P. Giri, Tetrahedron Lett., 935 (1987); A. P. Schaap, T. S. Chen, R. S. Handley, R. DeSilva, and B. P. Giri, Tetrahedron Lett., 1155 (1987); and A. P. Schaap, M. D. Sandison, and R. S. Handley, Tetrahedron Lett., 1159 (1987)). To do this, new synthetic procedures to produce dioxetanes with several key features have been developed: (1) the stabilizing influence of spiro-fused adamantyl groups has been utilized to provide dioxetanes that have "shelf lives" of years at ambient temperature and (2) new methods for triggering the chemiluminescent decomposition of the stabilized dioxetanes have been developed.
The required alkenes have been prepared by reaction of adamantanone with aromatic esters of ketones using titanium trichloride/LAH in THF (A. P. Schaap, U.S. Pat. No. 4,857,652). This is the first report of the intermolecular condensation of ketones and esters to form vinyl ethers using these reaction conditions. Although McMurry had earlier investigated the intramolecular reaction of ketone and ester functional groups, cyclic ketones and not vinyl ethers were prepared by this method (J. E. McMurry and D. D. Miller, J. Amer. Chem. Soc., 105, 1660 (1983) and J. E. McMurry, Chem. Rev., 89, 1513 (1989)). ##STR2##
Photooxygenation of these vinyl ethers affords dioxetanes that are easily handled compounds with the desired thermal stability. For example, the dioxetane shown below exhibits an activation energy of 28.4 kcal/mol and a half-life at 25.degree. C. of 3.8 years. Samples of this dioxetane in o-xylene have remained on the laboratory bench for several months with no detectable decomposition. ##STR3##
However, the chemiluminescent decomposition of this dioxetane can be conveniently triggered at room temperature by removal of the silyl protecting group with fluoride ion to generate the unstable, aryloxide form which cleaves to yield intense blue light. The half-life of the aryloxide-substituted dioxetane is 5 seconds at 25.degree. C. The spectrum of the chemiluminescence in DMSO exhibits a maximum at 470 nm which is identical to the fluorescence of the anion of the ester cleavage product (methyl 3-hydroxybenzoate) and the fluorescence of the spent dioxetane solution under these conditions. No chemiluminescence derived from adamantanone fluorescence appears to be produced. The chemiluminescence quantum yield for the fluoride-triggered decomposition measured relative to the luminol standard was determined to be 0.25 (or a chemiluminescence efficiency of 25%). Correction for the fluorescence quantum yield of the ester under these conditions (.O slashed..sub.F =0.44) gave an efficiency for the formation of the singlet excited ester of 57%, the highest singlet chemiexcitation efficiency yet reported for a dioxetane prepared in the laboratory. ##STR4##
2. Enzymatic Triggering of Stabilized 1,2-Dioxetanes. Biological assays such as immunoassays and DNA hybridizations involving enzymes utilized a wide variety of substrates which either form a color (chromogenic) or become fluorescent (fluorogenic) upon reaction with the enzyme. As part of the investigation of triggering methods, the first dioxetanes which can function as chemiluminescent enzyme substrates have been discovered (A. P. Schaap, U.S. patent application Serial No. 887,139; A. P. Schaap, R. S. Handley, and B. P. Giri, Tetrahedron Lett., 935 (1987); A. P. Schaap, T. S. Chen, R. S. Handley, R. DeSilva, and B. P. Giri, Tetrahedron Lett., 1155 (1987); A. P. Schaap, M. D. Sandison, and R. S. Handley, Tetrahedron Lett., 1159 (1897) and A. P. Schaap, Photochem. Photobiol., 47S, 50S (1988)). Use of these peroxides in biological systems requires dioxetanes which are thermally stable at the temperature of the enzymatic reaction and do not undergo rapid spontaneous decomposition in the aqueous buffers. The spiro-fused adamantyl dioxetanes described in the previous section meet these requirements. Dioxetanes have been prepared which can be enzymatically modified to generate the aryloxide form. Decomposition of this unstable intermediate provides the luminescence. Dioxetanes have been synthesized which can be triggered by various enzymes including aryl esterase, acetylcholinesterase, alkaline phosphatase and beta-galactosidase. ##STR5##
For example, enzymatic triggering by alkaline phosphatase has been observed with the phosphate-substituted dioxetane shown above derived from 3-hydroxy-9H-xanthen-9-one and adamantanone. The dioxetane is thermally stable with an activation energy of 30.7 kcal/mol and a half-life at 25.degree. C. of 12 years. The dioxetane is not only stable in organic solvents but also shows very slow spontaneous decomposition in aqueous buffers. Triggering experiments were conducted using alkaline phosphatase from bovine intestinal mucosa and the phosphate-protected dioxetane at pH 10.3 in 0.75M 2-amino-2-methyl-1-propanol buffer. The total light emission was found to be linearly dependent on the dioxetane concentration. The rate of decay of the emission is a function of enzyme concentration while the total light emission is independent of the enzyme concentration. The chemiluminescence spectrum for the phosphatase-catalyzed decomposition was obtained at room temperature in the buffer solution. A comparison of this chemiluminescence spectrum with the fluorescence spectrum of the spent reaction mixture and the fluorescence spectrum of the hydroxyxanthone cleavage product in the buffer indicates that the emission is initiated by the enzymatic cleavage of the phosphate group from the dioxetane to yield the unstable aryloxide dioxetane which generates the singlet excited anion of hydroxyxanthone. ##STR6##
Phosphatase triggering experiments have also been conducted using the phosphate-protected dioxetane shown above derived from methyl 3-hydroxybenzoate and adamantanone with alkaline phosphatase at pH 9.6 in 0.75M 2-amino-2-methyl-1-propanol buffer. Addition of the enzyme to a 10.sup.-4 M solution of dioxetane results in chemiluminescence emitted over several minutes. As a result of the very low background luminescence from slow hydrolysis of the dioxetane in the buffer, less than 10.sup.-18 mol (1 attomol) of alkaline phosphatase can be detected in the presence of enhancers (A. P. Schaap, H. Akhavan and L. J. Romano, Clin. Chem., 35, 1863 (1989) and A. P. Schaap, U.S. patent applications Ser. Nos. 07/224,681 and 07/317,585).
3. Chemiluminescent Direct Labels for Biological Assays.
(a) Luminol and isoluminol. The aminophthalhydrazides, luminol and isoluminol react with H.sub.2 O.sub.2 and a peroxidase enzyme catalyst under basic conditions with emission of light. The reaction is also catalyzed by small amounts of several metal ions including Fe(III), Cu(II) and Cr(III). The first chemiluminescent immunoassay using luminol as a label was reported by Schroeder for an assay of biotin. (H. R. Schroeder, P. O. Vogelhut, R. J. Carrico, R. C. Boguslaski, R. T. Buckler, Anal. Chem. 48, 1933 (1976)). Several applications of the use of luminol derivatives as labels have been reported since then (H. R. Schroeder in Luminescent Immunoassays: Perspectives in Endocrinology and Clinical Chemistry, M. Serio and M. Pazzagli, Eds., Raven Press, New york, pp 129-146 (1982); M. Pazzagli, G. Messeri, A. L. Caldini, G. Monetti, G. Martinazzo and M. Serio, J. Steroid Biochem., 19, 407 (1983); Bioluminescence and Chemiluminescence New Perspectives, J. Scholmerich, et al., Eds., J. Wiley & Sons, Chichester (1987)). Various enhancers have also been employed in conjunction with the use of luminol to increase the intensity of light emitted. These include D-luciferin (T. P. Whitehead, G. H. Thorpe, T. J. Carter, C. Groucutt and L. J. Kricka, Nature, 305, 158 (1983)) and p-iodophenol (G. H. Thorpe, L. J. Kricka, S. B. Mosely and T. P. Whitehead Clin. Chem., 31, 1335 (1985)). ##STR7##
(b) Acridinium and phenanthridinium esters. Aromatic esters of N-methyl acridinium and phenanthridinium carboxylic acid undergo a chemiluminescent reaction upon treatment with H.sub.2 O.sub.2 and a base. The stability of these esters decreases markedly at high pH. Incorporation of a linker group R on the aromatic moiety as shown below allows attachment of the chemiluminescent label to antigens, antibodies or solid supports. This work has been reported in a patent (F. McCapra, D. E. Tutt, R. M. Topping, British Patent No. 1,461,877 (1977)) and in the following papers (I. Weeks, I. Beheshti, F. McCapra, A. K. Campbell, J. S. Woodhead, Clin. Chem., 29, 1474 (1983); I. Weeks, A. K. Campbell, J. S. Woodhead, Clin. Chem., 29, 1480 (1983)). These compounds are used as labels in commercial immunoassay kits for the detection of TSH (LumaTag.TM.; London Diagnostics, Inc.,; Eden Prairie, Minn.) and free thyroxin (Magic Lite System; Ciba-Corning Diagnostics Corp.; Medfield, Mass.). This technology has also been used to label DNA probes (L. J. Arnold, Jr., P. W. Hammond, W. A. Wiese, N. C. Nelson, Clin. Chem., 35, 1588 (1989)). ##STR8##
(c) Adamantylideneadamantane dioxetanes for thermochemiluminescent detection. Derivatives of adamantylideneadamantane dioxetane have been prepared by Wynberg and co-workers for the direct labeling of antibodies and proteins. (J. C. Hummelen, T. M. Luider, H. Wynberg, Pure Appl. Chem., 59, 639 (1987); J. C. Hummelen, T. M. Luider, H. Wynberg, "Thermochemiluminescent Immunoassays", in Complementary Immunoassays, W. P. Collins, Ed., Wiley and Sons, New York, p. 191 (1988); H. Wynberg, E. W. Meijer, J. C. Hummelen, 37 1,2-Dioxetanes as Chemiluminescent Probes and Labels" in Bioluminescence and Chemiluminescence, M. A. DeLuca, W. D. McElroy, Eds., Academic Press, New York, p. 687 (1981)). Detection of chemiluminescence requires that the labeled analyte be immobilized on a plastic disk and heated to 250.degree. to 300.degree. C. These extreme temperatures are incompatible with the development of a homogeneous immunoassay system which is performed in aqueous solution. Since the chemiluminescence quantum yield from the thermolysis of adamantylideneadamantane dioxetane is quite low (.O slashed..sub.CL =10.sup.-4) the technique of energy transfer to a highly fluorescent acceptor must be employed to achieve higher sensitivity of detection. This requires that a second label, a fluorescer, also be chemically attached either to the analyte or to the first label (J. C. Hummelen, T. M. Luider, H. Wynberg, Methods in Enzymology, 133, 531 (1986)). Reaction R groups which were used to chemically bind the dioxetane to proteins included those shown below. ##STR9##
4. Labeling Procedures. A wide variety of procedures for chemically binding labels to organic and biological molecules are described in the literature (see, for example: L. J. Kricka, Ligand-Binder Assays, Marcel Dekker, Inc., New York, 1985, pp. 15-51 and M. Z. Atassi, "Chemical Modification and Cleavage of Proteins," Chapter 1 in Immunochemistry of Proteins, Vol. 1, Plenum Press, New York, 1977, pp. 1-161, and references therein). Antibodies and proteins are conveniently labeled by reaction of certain nucleophilic groups present in proteins (--SH, --OH, --NH.sub.2, --COOH) with labels bearing chemically reactive groups such as those shown below in Table 1. Appropriately functionalized nucleic acids and DNA probes can also be labeled by synthesizing amine or thiol-containing nucleic acids and reacting these molecules with the corresponding reactive group on the dioxetane. Alternatively, nucleic acids (primarily oligonucleotides) can be linked to dioxetanes by the reaction of a hydroxyl with an activated phosphoramidite. In this case the phosphoramidite could be positioned on the nucleic acid and the hydroxyl placed on the dioxetane or vice versa. Other types of molecules which can be labeled include enzymes, protein antigens, haptens, steroids, carbohydrates, fatty acids, prostaglandins, thromboxanes, leukotrienes, nucleosides and nucleotides.
TABLE 1 ______________________________________ Reactive Groups for Chemical Binding of Labels to Organic and Biological Molecules 1) Groups which are reactive towards amines (NH.sub.2). ##STR10## ##STR11## ##STR12## ##STR13## ##STR14## ##STR15## ##STR16## ##STR17## ##STR18## ##STR19## ##STR20## ##STR21## ##STR22## ##STR23## ##STR24## ##STR25## ##STR26## ##STR27## 2) Groups which are reactive towards thiols (SH). ##STR28## CH.sub.2 -Halogen ##STR29## ##STR30## ##STR31## ##STR32## ##STR33## SSR ##STR34## 3) Groups which are reactive towards carboxylic acids (CO.sub.2 H). NH.sub.2 OHNHNH.sub.2 ______________________________________
Bifunctional coupling reagents may also be used to couple labels to organic and biological molecules with moderately reactive groups (see L. J. Kricka, Ligand-Binder Assays, Marcel Dekker, Inc., New York, 1985, pp. 18-20, Table 2.2 and T. H. Ji, "Bifunctional Reagents," Methods in Enzymology, 91, 580-609 (1983)). There are two types of bifunctional reagents, those which become incorporated into the final structure and those which do not and serve only to couple the two reactants. Fluorescein can be used to form a complex with anti-fluorescein. Additionally, psoralen can be used to covalently bind to DNA.
Physical binding or complex formation of a chemiluminescent dioxetane to a molecule of interest may be accomplished as shown below by chemical bonding of the label to a molecule of biotin (vitamin H) and chemical bonding of the organic or biological molecule of interest to either avidin or streptavidin. The latter two compounds are bacterial proteins with four high-affinity binding sites for biotin (R. M. Buckland, Nature, 320, 557-558 (1986) and references therein).